Glycans are widely distributed in biological systems in the free state as well as conjugated forms as parts of glycoproteins, glycolipids, and proteoglycans. They are involved in a wide range of biological and physiological processes including recognition, regulatory functions, cellular communication, gene expression, stability and activity of proteins therapeutics, cellular immunity, growth and development. Biological and physiological functions of glycans are often dependent on the structure and types of oligosaccharides attached to the proteins or lipids. The structures of glycans are quite diverse and complex due to post-translational modifications and physiological conditions. Thus, it is highly challenging to analyze comprehensive glycan profiles (i.e. glycome) and determine the structure of glycans for clinical use
HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) was the first routine assay method developed for glycan analysis. This ion chromatography (IC) method can separate carbohydrates via specific interactions between the hydroxyl groups of glycans and chromatographic medium at high pH. The glycans are chromatographed as anionic species and interact with the column based on glycan charge, size, composition, isomers and linkage(s). This analytical method provides a profile of the overall glycosylation or oligosaccharide population present on a product, or at a specific glycosylation site, which can be used for batch comparison studies. However, a significant disadvantage of HPAEC-PAD separation of glycans is the need to use a relatively high salt concentration. In such a case, a desalter column is often used so that the mobile phase is compatible with mass spectrometry.
Various modes of HPLC separation have been developed for the analysis of glycans, including normal phase or hydrophilic interaction (HILIC), ion-exchange and reversed-phase. Because glycans are highly hydrophilic and polar substances, HILIC is extensively used for glycan analysis. The separation columns are typically amide, amine or zwitterionic-based packing materials. The amide HILIC column separates glycans mainly by hydrogen bonding, resulting in size and composition based separation. However, no charge based separation can be achieved with this approach. Both amine-based and zwitterionic columns show ionic interactions with charged glycans (native or labeled) and, although they separate glycans based on charge, they provide relatively low resolution between glycans of the same charge (e.g., polarity and/or magnitude of charge). In addition, the inherently strong ionic interactions between multiply charged glycans and the stationary phase often requires a high eluent buffer concentration, adversely affecting MS sensitivity.
A few reports have described the use of amino or quaternized amine columns as anion-exchange/HILIC mixed-mode columns for glycan analysis (Ruhaak et al., Anal. Bioanal Chem (2010) 397:3457-3481; Anumula et al., Anal Biochem (2000) 283:17-26; and Neville et al., J Proteome Res (2009) 8:681-687). However, the amino or quaternized amine columns referenced above have only one mode, which is an ion exchange mode, and thus, are not mixed mode. In addition, these columns have only one type of chromatographic functional group, which in this case is either an amino or quaternized amine. The above references do not describe separation conditions over a pH range where the amino group would have a neutral charge (i.e., unprotonated). Note that a quaternized amine cannot have a neutral charge even at high pH. A protonated amine group will act as anion exchange group and not as a HILIC group. It should be noted that although increasing retention times with increasing solvent concentration provides evidence of a HILIC interaction, it also provides evidence of other retention modes such as ion exchange and the interaction of salts with zwitterionic modes. As such, increasing retention times with increasing solvent concentration does not by itself indicate a HILIC interaction. Amino and quaternized amine columns suffer from the disadvantages of having a long retention time (e.g., >60 minutes) and requiring relatively high salt concentrations. In general, relatively high salt concentrations interfere with the detection of glycans with mass spectrometry.
Reports have described the use of zwitterionic sulfobetaine columns as a zwitterionic ion chromatography (ZIC)/HILIC mixed-mode columns for glycan analysis (Ruhaak et al., Anal. Bioanal Chem (2010) 397:3457-3481; Takegawa et al., Journal of Chromatography A, (2006) 1113:177-181; and Takegawa et al., J Sep Sci (2006) 29:2533-2540). However, the zwitterionic columns referenced above have only one mode, which is a zwitterionic mode based on the sulfobetaine stationary phase, and thus, are not mixed mode. In addition, these columns have only one type of chromatographic functional group, which in this case is a zwitterionic sulfobetaine group. Such sulfobetaine groups are net neutral over a very broad pH range, interacting via an electrostatic mechanism with salts in an ionic strength dependant manner, which is different than an uncharged HILIC mechanism. It should be noted that although increasing retention times with increasing solvent concentration provides evidence of a HILIC interaction, it also is indicative of other retention modes such as ion exchange and the interaction of salts with zwitterionic modes. As such, increasing retention times with increasing solvent concentration does not by itself indicate a HILIC interaction. The zwitterionic phase column suffers from the disadvantage of showing relatively poor resolution for a group of glycans having the same charge.
It is generally very desirable to detect and characterize glycan analytes in the eluate by using mass spectrometry, however, the art clearly teaches that operating under conditions in which the anion-exchange phase is highly ionized is unacceptable when combined with mass spectroscopic detection. The zwitterionic mode operates via a salt exchange retention mechanism, which is inherently less selective than ion exchange. High solvent concentrations simply increase the strength of the salt exchange interactions.
Applicant believes that there is a need for new multimodal chromatographic media and methods of analyzing glycans using these media that would ideally provide high resolution between different glycans, unique selectivity based on size, composition, structure (e.g., isomerism, linkages), and/or charge. Further, Applicant also believes that if the glycans eluted from the media could be detected by standard methodology (e.g., mass spectrometry, fluorescence detection) with no, or minimal, clean up or purification post-analysis and pre-detection (e.g., fluorescent tagging), this would greatly simplify and facilitate glycan analysis. The present invention provides such chromatographic media and methods of using them.